This proposal is to study a candidate virulence gene and its role in H influenzae disease. The specific aims of this project are: 1) define the complete nucleotide sequence of hitB, a homolog of the E coli hemolysin transport gene hlyB, in Haemophilus influenzae type b (Hib) and nontypable strains (NTHi); 2) use a genetic approach and protein analysis strategy to identify the putative protein(s) transported by HitB; 3) characterize hit B function in H. influenzae using hitB mutants and screening of clinical isolated. Preliminary nucleotide sequence data has identified a region homologous to hlyB on subclones containing Hib chromosomal DNA. In addition, Southern analysis suggests that this region is also present in NTHi. HlyB is associated with hemolysin transport in E coli, and analogous proteins in the Pasteurellaceae family transport various cytotoxins of the RTX family. The genes involved in encoding the RTX toxin, RTX activators, and transporters of the toxin are classically organized in a cluster on the chromosome. This proposal will investigate the relationship of hitB with virulence factor transport in H. influenzae. Nucleotide sequence analysis will be performed to define the boundaries of hitB in Hib. The homologous region will be cloned from NTHi and the sequence of the hitB gene in NTHi will be determined. Identification of the protein(s) transported by HitB will use several approaches. The region flanking hitB will undergo sequence analysis to determine the presence of genes encoding transported protein candidates. Protein analysis of various cell fractions of wild type Hib in comparison with hitB mutants will be used to identify differential protein transport. In addition, the HitB protein will be utilized in binding assays to identify and purify a candidate protein transported by HitB. An RTX cytotoxin gene or protein will be screened for using Southern blots or immunoblots. Finally hitB function will be characterized by examining the phenotypic effects produced by mutations in hitB on growth, piliation, hemagglutination and cytotoxic activity, and by screening for HitB expression in H. influenzae associated with disease and with normal flora.